PROJECT SUMMARY/ABSTRACT Gastroparesis from diabetes mellitus is associated with significant morbidity and represents a major health care burden. Therefore, finding effective treatment options for this disorder is an imminent need. A critical barrier to achieving this goal is the incomplete understanding of the specific molecular mechanisms underlying the cellular-level pathogenesis of impaired gastric motor functions. Previous research has identified the key cellular targets of diabetes in gastrointestinal muscle layers including macrophages, ICC and enteric nerves. Neurons expressing nitric oxide (NO) synthase 1 (Nos1) are the component of the enteric nervous system most consistently affected in gastroparesis; and there is strong evidence that depletion of Nos1+ neurons and the consequent impaired nitrergic inhibitory neurotransmission are pathogenetically significant. Therefore, the overall goal of this project is to determine the molecular mechanisms controlling Nos1+ neuron populations in diabetic gastroparesis. Recent data from the Diabetes Control and Complications Trial (DCCT), the follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) study and Project 3 indicate that successful prevention of microvascular complications and gastroparesis depends on glycemic control in the distant past and raised the possibility that this ?metabolic memory? is encoded by epigenetic control of gene transcription. Therefore, based on preliminary data and leveraging our relevant expertise and research arsenal we hypothesize that epigenetic dysregulation of Nos1 transcription underlies persistent reduction in Nos1 expression in diabetes. We also propose that reversal of this repression would not only recover Nos1+ neurons that have downregulated Nos1 transcription from diabetes but also replace lost cells by recruiting, via epigenetic reprogramming, new Nos1 neurons from a pool of existing cells with developmentally repressed Nos1 expression. Our first specific aim 1 is to determine the therapeutic potential of inhibiting the epigenetic silencers Ezh2 and histone deacetylases to restore the pool of Nos1+ neurons in diabetic gastroparesis. Our second specific aim is to determine the therapeutic potential of facilitating bone morphogenetic protein-induced histone acetylation by upregulating hypoxia-inducible factor 1 ? to further increase Nos1-expressing neurons and restore gastric function. We will combine genetic lineage tracing, in-vivo genome editing, RNA interference and recombinant DNA techniques with quantitative flow cytometry, cell sorting, transcriptomics, multi- parameter epigenomics and integrated bioinformatic analyses, as well as confocal imaging and physiological studies to address our aims. This project also has significant translational focus and potential due to the testing, in preclinical models, three classes of drugs already approved for human use thus setting the stage for future clinical trials in collaboration with Project 3. The concept of manipulating enteric neuron populations via epigenetic reprogramming of cells with persistently repressed transcriptional programs will change how we think about neuronal plasticity in health and disease.